Enhanced immunization and suppression of oral tolerance

ABSTRACT

This invention provides for methods of immunizing mammals against tumors expressing a carcinoembryonic antigen using a carcinoembryonic immunogen modified to lack cellular immunosuppressive activity or CD1d binding. The invention further provides for use of a carcinoembryonic immunogen having a region recognized by antibody B9 or L12 modified to inactivate cellular immunosuppressive activity. Methods are also provided for modifying the immunogen by deleting, altering, mutating or truncating the immunosuppressive region. The invention also provides for enhancing cellular immunogenicity of an orally delivered immunogen by co-administration of an agent capable of inhibiting the immunosuppressive activity of carcinoembryonic antigen. Further methods are provided for creating a patient having a carcinoembryonic-antigen-family-member-expressing tumor by immunizing the patient with the carcinoembryonic-antigen-family-member lacking the immunosuppressive region. Pharmaceutical compositions for eliciting an effective immune response to the carcinoembryonic antigen are also provided.

FIELD OF THE INVENTION

This invention relates to methods for immunizing mammals against tumorsexpressing a carcinoembryonic antigen by immunizing the mammal with acarcinoembryonic antigen that has been modified to lackimmunosuppressive activity. The invention further relates to enhancementof cellular immunogenicity of orally-delivered immunogens byco-administration of the immunogen with agents capable of inhibiting theimmunosuppressive activity of carcinoembryonic antigen, methods oftreating patients having a carcinoembryonic antigen family memberexpressing tumor, and pharmaceutical compositions for effectivelyeliciting an immune response to a carcinoembryonic antigen familymember.

BACKGROUND OF THE INVENTION

The nature of the immune response in the intestine is one of suppressionor controlled inflammation. Several groups have suggested that this typeof response is dictated by the nature of the microenvironment in whichthe mucosa-associated lymphoid tissue resides. Several factors come intoplay to promote this suppressed state: regulatory T cells, poorlyreactive macrophages, unusual T lymphocyte populations (intraepithelialand lamina propria lymphocytes) and unique antigen presenting cells.Included in this later population are intestinal epithelial cells.Evidence has been provided for such functional properties in both rat,mouse and man. In rat and man, antigens presented by these cells resultin the selective activation of CD8⁺ suppressor T cells.

Two cell surface molecules involved in this interaction have beenidentified: the nonclassical class I molecule CD1 d and a surfaceglycoprotein recognized by two anti-epithelial cell monoclonalantibodies (mAbs), B9 and L12, called gp180. These two mAbs block theselective proliferation of CD8⁺ T cells and inhibit the phosphorylationof the CD8 associated kinase p56 1ck in IEC: T cell co-cultures.Purified gp180 (mAb B9 affinity purified material) binds to CD8,activates p56 1ck and forms a complex with CD1d.

It is towards the identification and further characterization of gp180and its exploitation for immunomodulatory purposes that the presentinvention is directed. SUMMARY OF THE INVENTION

In a first aspect, the present invention is directed to a method foreffectively immunizing an animal against acarcinoembryonic-antigen-family-member-expressing tumor by at leastimmunizing the animal using an immunogen consisting of acarcinoembryonic antigen family member lacking cellularimmunosuppressive activity. The carcinoembryonic antigen family memberlacking cellular immunosuppressive activity may be a carcinoembryonicantigen (CEA) family member which has an alteration or deletion of aregion that is recognized by monoclonal antibody B9, or it may beGYSWYKGERVDGNRQII (SEQ ID NO:1), WYKGERV (SEQ ID NO:2) or YKGERVD (SEQID NO:3). The lacking of the cellular immunosuppressive region oractivity may be achieved by altering, deleting, replacing, or otherwisemodifying one or more amino acids in the cellular immunosuppressiveregion of the CEA family member molecule, such that the altered proteinlacks the ability to suppress an immune response or induce suppressor Tcells capable of suppressing an immune response. The suppression may besuppression of an antibody response, the suppression of a cytotoxiccellular response, or suppression of both responses. In a preferredembodiment, the carcinoembryonic antigen family member iscarcinoembryonic antigen. The animal is preferably a mammal, mostpreferably a human.

The invention is also directed to compositions and pharmaceuticalcompositions comprising one or more CEA family members with an alteredcellular immunosuppressive region such that the composition when used asa vaccine or immunogen is capable of eliciting an effective humoraland/or cellular immune response against the CEA family member, withoutthe immunosuppressive activity contributed by the cellularimmunosuppressive region of the CEA family member. Alterations of theregion are exemplified above, but are not so limiting. Preferably, theCEA family member is carcinoembryonic antigen. The composition mayfurther comprise a pharmaceutically-acceptable carrier, excipient, ordiluent.

In a further aspect, the invention is directed to a method foreffectively immunizing an animal against a tumor that is expressing acarcinoembryonic antigen family member by at least immunizing the animalwith a carcinoembryonic antigen family member together with an agentcapable of inhibiting a cellular immunosuppressive region of thecarcinoembryonic antigen family member. The agent may be, for example,an inhibitor of the engagement of CD8 with a cellular immunosuppressiveregion of the carcinoembryonic antigen family member, such as amonoclonal antibody such as B9 or L12, or a CD8 peptide or CD8 fusionpeptide capable of binding to the immunosuppressive epitope on a CEAfamily member. It also may be an inhibitor of the engagement of CD1dwith a region of the carcinoembryonic antigen family member.Non-limiting examples of a CD8 peptide or fusion peptide include aCD8-Fc fusion peptide or a CD8 peptide comprising an epitope recognizedby any one of antibodies OKT8B, OKT8E, OKT8F or OKT8I1. The monoclonalantibodies are preferably humanized monoclonal antibodies. Preferablythe animal is a mammal, most preferably a human.

In still yet a further aspect, the invention is also directed to amethod for enhancing the immunogenicity of an orally-delivered immunogenin an animal by at least co-administering the orally-delivered immunogenwith an agent capable of inhibiting the cellular immunosuppressiveactivity of a carcinoembryonic antigen family member. In one embodiment,the agent is a monoclonal antibody capable of disrupting the engagementof a carcinoembryonic antigen family member with CD8 or CD1d, such asbut not limited to monoclonal antibodies B9 or L12, or a CD8 peptide,CD8 fusion peptide, CD1d peptide or CD1d fusion peptide capable ofbinding to the immunosuppressive epitope on a CEA family member, or aCD1d peptide or CD1d fusion peptide capable of inhibiting theinteraction of a CEA family member with CD1d. Non-limiting examples ofsuch CD8 peptides and CD8 fusion peptides include a CD8-Fc fusionpeptide or a CD8 peptide comprising an epitope recognized by any one ofantibodies OKT8B, OKT8E, OKT8F or OKT8I1. A humanized monoclonalantibody is preferred. The enhanced immunogenicity may be a humoral,cellular, or both responses.

In yet still a further aspect of the invention, a method is provided forsuppressing a humoral or cellular response to an antigen by providing atthe site of the antigen a carcinoembryonic antigen, a carcinoembryonicfamily member, or a fragment, insertion, or fusion polypeptide of CEA ora CEA family member comprising the cellular immunosuppressive region,such that any potential immune response elicited by the antigen may besuppressed by the presence of the activity of the cellularimmunosuppressive region of CEA or a CEA family member. Such a regionis, for example, the region of CEA depicted in SEQ ID NO:1, or is aregion that is recognized by monoclonal antibodies B9 or L12. In afurther embodiment of this aspect of the invention, a protein comprisingthe cellular immunosuppressive region of a CEA family member may beintroduced by genetic means into a site within the body, such as acellular mass or tissue, such that expression and secretion of theprotein comprising the cellular immunosuppressive region abrogates animmune response. This aspect of the invention is preferably applied toautoimmune diseases, such as but not limited to inflammatory boweldisease, ulcerative colitis, Crohn's disease, lupus, psoriasis, asexamples of diseases in which an inappropriate immune response to anendogenous or exogenous antigen is pathogenetic.

It is thus an object of the invention to provide a method foreffectively immunizing an animal against acarcinoembryonic-antigen-family-member-expressing tumor by immunizingthe animal using an immunogen consisting of the carcinoembryonic antigenfamily member lacking an active cellular immunosuppressive region orCD1d binding. The active cellular immunosuppressive region includes amonoclonal antibody B9- or L12-recognizing region.

In a preferred embodiment, the active cellular immunosuppressive regionis SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. The aforementionedimmunogen may have a deletion, alteration; mutation, or truncation ofthe active cellular immunosuppressive region which abrogates itscellular immunosuppressive activity. The cellular immunosuppressiveactivity may be directed to a humoral, or cellular response or both.

The carcinoembryonic antigen family member preferably iscarcinoembryonic antigen.

It is another object of the invention to provide a method foreffectively immunizing an animal against a tumor expressing acarcinoembryonic antigen family member by at least immunizing the animalwith the carcinoembryonic antigen family member together with an agentcapable of inhibiting a cellular immunosuppressive region of thecarcinoembryonic antigen family member. The agent may be a ligand of thecellular immunosuppressive region, such as but not limited to amonoclonal antibody such as B9 or L12. The agent may be an inhibitor ofthe engagement of CD8 with a cellular immunosuppressive region of acarcinoembryonic antigen family member, or of the engagement of CD1dwith a cellular immunosuppressive region of a carcinoembryonic antigenfamily member. Preferably, the carcinoembryonic antigen family member iscarcinoembryonic antigen (CEA), and the mammal a human.

It is a further object of the invention to provide a method forenhancing cellular immunogenicity of an orally-delivered immunogen in ananimal by co-administering with the orally-delivered immunogen an agentcapable of inhibiting the cellular immunosuppressive activity of acarcinoembryonic antigen family member. The agent may be a ligand of thecarcinoembryonic antigen family member capable of disrupting theengagement of the carcinoembryonic antigen family member with CD8 orCD1d, such as but not limited to a monoclonal antibody, such as B9 orL12. The agent is an inhibitor of the engagement of CD8 of CD1d with acellular immunosuppressive region of the carcinoembryonic antigen familymember. Preferably, the carcinoembryonic antigen family member iscarcinoembryonic antigen, but it is not so limiting.

It is another object of the invention to provide a method for treating apatient having a carcinoembryonic-antigen-family-member-expressing tumorby at least immunizing the patient with an immunogen consisting of thecarcinoembryonic antigen family member lacking a cellularimmunosuppressive region. The CEA family member may be lacking a B9/L12epitope having an alteration, deletion, mutation, or other change whichabrogates the cellular immunosuppressive activity of the region.

It is yet another object of the invention to provide a method forsuppressing a humoral or cellular immune response in a mammal to anantigen by at least administering to a site of the humoral or cellularresponse in the mammal an agent comprising a cellular immunosuppressiveregion of a carcinoembryonic antigen family member. The agent comprisinga cellular immunosuppressive region of a carcinoembryonic antigen familymember may be carcinoembryonic antigen, a carcinoembryonic antigenfamily member, or a fragment or fusion polypeptide of either of theforegoing comprising a cellular immunosuppressive region. Theadministering may be by introducing a vector capable of inducingexpression of said agent in cells of said mammal. Preferably, the mammalis a human, and the carcinoembryonic antigen family member iscarcinoembryonic antigen. This method is useful for the treatment ofchronic inflammatory and autoimmune disorders.

It is still yet another object of the invention to providepharmaceutical compositions for eliciting an effective immune responseto a carcinoembryonic antigen family member comprising an immunogenconsisting of the carcinoembryonic antigen family member lacking anactive cellular immunosuppressive region. The cellular immunosuppressiveregion may be a monoclonal antibody B9- or L12-recognizing region, suchas SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. The lacking may be achievedby a deletion, alteration, mutation, or truncation of the activecellular immunosuppressive region. Preferably, the carcinoembryonicantigen family member is carcinoembryonic antigen.

These and other aspects of the invention will be apparent from thefollowing figures and the ensuing detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the homology between a sequence of an antigen that binds tomonoclonal antibody B9 and various CEA family members.

FIG. 2 shows that transfected, GFP-CEA-expressing 293T cells arerecognized by monoclonal antibody B9.

FIG. 3A-B show that mAb B9 and mAb L12 reactivity were both isolated tothe N domain, specifically within the loop formed by the sequenceGYSWYKGERVDGNRQII (SEQ ID NO:1) (FIG. 3A); and that a sugarless mutanttransfectant was still capable of being stained with mAb B9 (FIG. 3 b).

FIG. 4 shows that CEA binds to CD8α chains in that only CD8α but not CD4transfected hybridoma cells were capable of absorbing B9 reactivematerial.

FIGS. 5A-B shows that mAb B9 recognized other CEA family members:CEACAM6(NCA), CEACAM1-4L and CEACAM8 (CGM6) transfectants. The CEAsubfamily members recognized by B9 share the B9 epitope in the N domaineither exactly or with minor modification.

FIGS. 6A-B show, using increasing amounts of purified gp180 (CEA) addedto a CTL assay testing alloreactive CTL that, unlike mAbs to MHC class Ior CD8 α which both inhibited CTL activity, gp180 induced only a modestdecrease (<15% in 3 experirnents) in killing (FIG. 6A). In contrast,when varying concentrations of purified gp180 were added to cultureswhere CTLs were induced, there was complete inhibition of generation ofCTLs (FIG. 6B).

FIG. 7A-B shows that an A2 expressing B cell line and autologous MNCprimed for a CTL response whereas the A2 expressing, gp180 expressingHT29 cells did not.

FIGS. 8A-B shows that co-culture of PB T cells with allogeneic adherentcells (DCs, monocytes) resulted in both phosphorylation of PI3K and Vav.Addition of gp180 inhibits PI3K phosphorylation and enhances thekinetics of phosphorylation of vav.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors, in further studies of the intestinal epithelialcell glycoprotein gp180, have identified it as the oncofetal antigenknown as carcinoembryonic antigen, or CEA. The attribution of thecharacteristics of gp180 to CEA, and the recognition that CEA isnaturally expressed on intestinal epithelial cells, offers several newmodalities for both improvements of mucosal vaccination in general, aswell as a means for improving the immunogenicity of CEA, a popular yetseemingly recalcitrant target for immunotherapy for cancers thatoverexpress CEA. Moreover, as other CEA family members share commonaspects with gp180, now recognized to be CEA, immunotherapeticapproaches using other CEA family members, such as cancer immunotherapy,may also be enhanced.

In the aspect of the invention directed to enhancing the immune responsespecifically to CEA and its family members, the invention hereinprovides an explanation for the poor immunogenicity elicited by CEA invarious cancer immunotherapy trials that have been performed to date,and offers a means for rectifying the poor immunogenicity. The inventorsherein have identified an epitope of CEA extending from amino acids 30to 46, GYSWYKGERVDGNRQII (SEQ ID NO:1), against which monoclonalantibodies raised thereto block the cellular immunosuppressive activityof CEA. Specifically, monoclonal antibodies B9 and L12, which recognizethe epitopes WYKGERV (SEQ ID NO:2) and YKGERVD (SEQ ID NO:3),respectively, when included in an IEC:T cell culture, block the cellularimmunosuppressive activity of CEA. Thus, elimination of theaforementioned epitope in vaccines or other immunogens against which animmune response to CEA is desired will avoid the cellularimmunosuppressive activity of CEA and allow an effective anti-CEAcellular response to be elicited. Such an effective CEA immunogen may beachieved using fragments of CEA exclusive of the aforementioned cellularimmunosuppressive region, or a fusion polypeptide which deletes thecellular immunosuppressive region from the CEA molecule.

The cellular immunosuppressive region of CEA or of a CEA family memberis defined herein as a region: 1) that binds to CD8 and alters signalingsuch that cytotoxic T cells are not generated and 2) that activatesregulatory or suppressor T cells which mediate immunosuppression. Thelatter activity is mediated by the interaction between a CEA familymember and the class lb molecule CD1d. Either or both of theseactivities may be suppressed, or enhanced, by the methods and agents ofthe invention.

The aforementioned strategies in achieving an effective cellular immuneresponse against CEA, for the treatment of CEA-expressing cancers, mayalso be translated to other CEA family members, such as NCA, CGM-2, CGM6and BGP, or, using newly-adopted nomenclature, CEACAM6, CEACAM7, CEACAM8and CEACAM1, respectively; or, to other molecules expressing the B9/L12epitopes described herein, for the treatment of cancers in which theseCEA family member proteins are expressed and are thus targets for cancerimmunotherapy.

In a further embodiment of the invention, agents and methods areprovided for the treatment of CEA-expressing tumors beyond the methodsdescribed above, in which a more effective CEA vaccine is provided. Theresults presented here provide an explanation for the immunosuppressionassociated with these tumors. Moreover, increased expression of CEA inconjunction with any other tumor antigen explains the failure of theimmune system to identify the appearance of such other tumor antigens.Thus, upon identification of the increased expression of CEA in anydysproliferative disease, a cause for general cellular immunosuppressionis identified, and therapies to enhance a cellular response in thepresence of the cellular immunosuppressive CEA may be initiated, asdescribed herein, to elicit an effective cellular response. As will beseen below, gp180/CEA does not inhibit the effector function ofcytotoxic T lymphocytes, so that if the therapies described hereinsuccessfully elicit a cellular immune response against the targetantigen, be it CEA or another antigen, the effector cells will carry outtheir cytolytic activity against cells expressing the antigen.

As alluded to above, the invention extends beyond therapeutic modalitiesto enhance the immunogenicity of CEA and avoid a cellularimmunosuppressive response. The identification of gp180 as CEA, incombination with the recognition that intestinal epithelial cells areantigen presenting cells, that CEA engages CD8 resulting in suppressionof a cytotoxic T cell response, and that CEA activates a T suppressorcell response by association of gp180/CEA with CD1d as described inCampbell et al., 1999, J. Biol. Chem. 274:26259-26265, incorporatedherein by reference in its entirety, provides a new means for enhancingthe immunogenicity, particular of a cellular response, against anyantigen, immunogen or vaccine that is presented by gp180/CEA-bearing or-expressing cells, such as intestinal epithelial cells. In a particularembodiment, such vaccines delivered mucosally, i.e., oral vaccines, maynot elicit a desirably robust cellular immune response thereto for thereasons mentioned above and the invention is directed to oralvaccination in conjunction with a means for blocking the cellularimmunosuppressive effects of gp180/CEA. While Applicants are not boundto theory, the finding of the immunosuppressive effects of gp180/CEA onCD8+ T cells and that CD1d restricted T cells inhibit other immuneresponses, the attendant inhibition of a cytotoxic T-cell response,and/or activation of T-suppressor cells, and their heretofore unknownand now surprising correlation with CEA, may be advantageously used inavoiding such immunosuppressive activities by the means describedherein. Such means, as mentioned above, include co-administration of anagent which blocks the cellular immunosuppressive region of CEA,antibodies which bind to and block the cellular immunosuppressiveactivity of CEA, and other agents which block signalling resulting fromthe engagement of CD8 or CD1d with the aforementioned region of CEA.These are merely non-limiting examples of methods and agents which maybe used to abrogate oral tolerance during desired immunizationprotocols.

Furthermore, in certain instances, immunosuppression is desirable, suchthat the cellular immunosuppressive region of a CEA family member is auseful activity. For example, in autoimmune diseases and other undesiredor inappropriately robust immune responses, methods for abrogating ahumoral and/or cellular immune response are desirable. For example, invarious inflammatory bowel diseases such as ulcerative colitis andCrohn's disease, as well as other localized and systemic autoimmunediseases including lupus and psoriasis, an undesired immune responseexists, and flare-up of the disease has an immunological basis. Inaccordance with the present invention, use of a CEA family member whichhas an active cellular immunosuppressive region, or a truncation, fusionpolypeptide, or other agent with the properties of the aforedescribedregion of various CEA family members is useful for local or systemictherapy, disease dependent, for abrogating a humoral or cellular immuneresponse. A congenital or acquired absence of CEA or CEA family memberis believed to promote chronic inflammation; gene therapy to theaffected cellular population or tissue provides a means for providing orincreasing the production of CEA or CEA family member and the attendantand desirable, in this instance, suppression of a humoral and/orcellular response.

The preferred carcinoembryonic antigen family member immunogens for usein a vaccine for eliciting a cellular response to the carcinoembryonicantigen family member are modified to lack an active region of thecarcinoembryonic antigen family member molecule wherein CD8 or CD1dengagement occurs. In the case of carcinoembryonic antigen, the area isshown generally in SEQ ID NO:1, and more specifically as SEQ ID NO:2,the epitope recognized by monoclonal antibody B9. Thus, a preferred CEAimmunogen is lacking SEQ ID NO:1 or SEQ ID NO:2. The lacking of theregion may be achieved by partial or total deletion, alteration of acritical amino acid which confers activity, substitution with anothersequence to preserve the desired immunogenicity of the remainder of themolecule, formation of a fusion polypeptide or truncated molecule, allof which abrogate the cellular immunosuppressive activity of the CEAfamily member. A skilled artisan, based on understanding the presentinvention and the immunological activity conferred by the region of CEAidentified herein, will readily design molecules without the activity.Corresponding deletions of related carcinoembryonic antigen familymembers are also embraced herein for use as immunogens or compositionsin vaccines for the aforementioned uses.

In the instance where the cellular immunosuppressive region is desired,agents such as the immunosuppressive peptide portion of the CEA familymember, or fusion polypeptides, may be provided which offer the activityof decreasing the immune response.

The foregoing modified carcinoembryonic antigen family member for use asa vaccine may be administered to an animal patient, preferably amammalian patient, and most preferably a human patient, by routinemethods known to elicit a cellular response for immunogens that normallyelicit a cellular response. A preferred but non-limiting route is oral,mucosal, intradermal, intramuscular or subcutaneous. The immunogens ofthe invention lacking the activity of the cellular immunosuppressiveregion elicit a therapeutic or protective cellular response and thus arecapable of an antibody or cytotoxic activity against cells expressingthe carcinoembryonic antigen family member.

In a further aspect, the cellular response of an orally- ormucosally-delivered immunogen other than a carcinoembryonic antigenfamily member may be enhanced by blocking the cellular immunosuppressiveactivity of a carcinoembryonic antigen family member, preferablycarcinoembryonic antigen, during antigen presentation. Agents whichdisrupt the engagement of the T cell CD8 molecule and the cellularimmunosuppressive region of the carcinoembryonic antigen family member,or block the association of the carcinoembryonic antigen family memberwith CD1d are co-administered to block the effect. The co-administeredagent may be administered by the same route or a different route thanthe immunogen, to provide effective blockage of the cellularimmunosuppressive effect. For example, a small-molecule antagonist ofthe carcinoembryonic antigen family member-CD8 interaction may beincluded in the oral vaccine, or may be administered parenterally to theindividual. In another embodiment, an agent capable of disrupting thesignalling events following carcinoembryonic antigen family member-CD8engagement may be administered.

As noted above, carcinoembryonic antigen family member deletion mutantsuseful for eliciting a cellular immune response are embraced herein.Such deletion mutants, or fragments lacking the cellularimmunosuppressive region, may be prepared by routine methods and thenscreened for successful elimination of cellular immunosuppression in anin-vivo assay. These assays for effective induction of a CTL responseare routine and readily undertaken by one skilled in the art.

Candidate patients or individuals for the therapeutic and protectivetherapies of the invention include animals, preferably mammals includinglivestock and domestic animals, and most preferably humans. While theexpression of various CEA family members among non-human mammalianspecies has been little studied, the teachings herein are applicablenotwithstanding the member(s) of the family expressed in a particularspecies. One target patient population for the methods herein is thatwith a carcinoembryonic antigen family member-expressing tumor in whichcontrol or eradication of the tumor is desired. Traditionally,carcinoembryonic antigen family member immunogens have been unsuccessfulat mounting an effective cellular response, now understood based on thepresent invention to be the presence of a cellular immunosuppressiveepitope amongst the carcinoembryonic antigen family member epitopes. Theepitope appears responsible for both suppression of CD8 signalling andthe induction of cytotoxic T cells, as well as activation of T cellswith suppressor activity. In another embodiment, the cellular immuneresponse to another antigen, such as an infectious disease immunogen orother cancer immunogen, particularly one delivered orally and presentedby mucosal cells, may be enhanced by blocking the cellularimmunosuppressive activity of the carcinoembryonic antigen familymember, particularly carcinoembryonic antigen, now known to be presentin intestinal (epithelial) cells and responsible based herein forreducing the effectiveness of immunogens presented thereby. Moreover,the CD8 T-suppressor cells generated by CEA are not target restricted.Vaccines comprising immunogens which enter the immune system through theintestinal epithelial cell are merely exemplary of such vaccines forwhich an enhanced cellular immune response is achievable by the methodsherein.

The invention is also directed to pharmaceutical compositions of theaforementioned CEA family member molecules lacking cellularimmunosuppressive activity, and to their use, for example, as aneffective immunogen for a CEA family member-expressing tumor. Examplesof such altered CEA family member molecules include CEA with an N domaindeletion, or a K35A substitution in the N domain, or a deletion ofGYSWYK or NRQII. These are described in Taheri, et al., 2000, J. Biol.Chem. 275:26935-43, which is incorporated herein by reference in itsentirety. These molecules may be formulated with appropriatepharmaceutically-effective carriers, excipients or diluent for local orsystemic administration, as appropriate for the prophylaxis or treatmentof a particular disease or condition.

As noted above, previous studies have suggested that normal intestinalepithelial cells can act as nonprofessional antigen presenting cells,selectively activating CD8⁺ suppressor T cells. As will be shown in theexamples below, an epithelial cell surface glycoprotein, initiallycalled gp180, recognized by monoclonal antibodies B9 and L12 wasdetermined to be critical to the activation of these regulatory T cells.Purification and sequence analysis of mAb B9 reactive material revealedsequence homology with carcinoembryonic antigen (CEA). Transfection ofCEA cDNA into CHO cells, 293T cells and F01 cells (melanoma line)conferred mAb B9 reactivity to these transfectants. Propertiespreviously attributed to gp180 such as CD8α binding and activation ofCD8 associated p561ck could be reproduced using purified CEA from thesetransfectants. The mAb B9 epitope was determined to be in the N domaincharacterized by the sequence GYSWYKGERVDGNRQII (SEQ ID NO:1). Thissequence is found with some variation in other CEA subfamily members aswell. These data suggest that CEA may be an important immunoregulatorymolecule expressed in the normal intestine and that increased expressionin malignancy may explain the immunosuppression associated with thesetumors.

EXAMPLE 1

Monoclonal Antibody B9 Affinity-Purified Material Bears SequenceHomology to CEA.

Monoclonal antibodies (mAb) B9 and L12 are described in Yio et al.,1997, J. Biol. Chem. 272:12786-92, incorporated herein by reference inits entirety. Two billion T84 cells were lysed with an octyl glucosidebased detergent buffer and passed over a mAb B9 affinity column. B9reactive material was then eluted with 2M glycine (pH 2.8) andconcentrated using YM 30 Centricon membranes. mAb B9 reactivity of theeluted and concentrated material was confirmed by Western Blot. Thepurified material was then subjected to N terminal sequencing by Edmandegradation. As seen in FIG. 1, over the first 25 amino acids, there was100% amino acid sequence homology with CEA and homologies ranging from65% to 96% with other CEA subfamily members.

EXAMPLE 2

CEA Transfectants Express the B9 Epitope

Given the homology to CEA, it was next determined whether this dominantmember of the CEA subfamily expressed the epitopes recognized by mAbs B9and L12. A CEA-GFP bicistronic construct was transfected into 293T andF01 cells. As seen in FIG. 2 GFP expressing 293T cells co-expressed amolecule recognized by mAb B9. This mAb did not recognize cells whichwere mock transfected or transfected with GFP alone. Similar data wereobtained using F01 cells and by FACS analysis of CHO cell transfectants(see FIG. 3). These data show that CEA expresses the B9 epitope.

Next, a series of CHO cells transfected with full-length CEA cDNA orcDNAs containing mutations in the N or C domain of CEA were analyzed. Asseen in FIG. 3 a, B9 reactivity was isolated to the N domain,specifically within the exposed loops formed by the sequenceGYSWYKGERVDGNRQII (SEQ ID NO:1). Destruction of this loop structureabrogated mAb B9 activity. mAb L12 recognizes a sequence overlapping theB9 epitope whereas other previously described anti-CEA mAbs B18 and A20recognize epitopes which are more C terminal to the B9 and L12 epitope.Interestingly, while the initial description of the B9 and L12 reactivematerial suggested that the epitope recognized by these mAbs requiredthe presence of N-linked sugars (epitope was removed by N-glycanasetreatment), a sugarless mutant transfectant was still capable of bindingmAb B9 (FIG. 3 b).

EXAMPLE 3

CEA Binds to CD8α Chains

CEA exists as a GPI anchored glycoprotein on the surface of IECs. Thismolecule can be cleaved using the enzyme PIPLC. CEA-GFP transfected 293T cells were treated with PIPLC and the liberated GPI anchored moleculeswere used for a series of absorption studies. Murine T cell hybridomastransfected with either human CD4 (3G4) or human CD8α (3G8) cDNA wereco-cultured with vector transfected or CEA transfected 293 T PIPLCtreated supernatants. Absorption of CEA by either CD4 or CD8 wasdetermined by immunoblotting with mAb B9. As seen in FIG. 4 only CD8αbut not CD4 transfected hybridoma cells were capable of absorbing the B9reactive material. These findings are consistent with functionalproperties previously ascribed to gp180.

EXAMPLE 4

mAb B9 Recognizes an Epitope on Other CEA Subfamily Members

Given the high homology of the N domain in a number of CEA subfamilymembers, it was next determined whether mAb B9 and L12 had a recognitionpattern restricted to CEA. CHO cells transfected with NCA (CEACAM6),BGPa (CEACAM1-4L), BGPx (CEACAM1-1L), CGM2 (CEACAM7), CGM6 (CC8) werestained with mAbs B9 and L12 and analyzed by flow cytometry. As seen inFIG. 5 a-b, mAb B9 recognized NCA, BGPa, and CGM6 transfectants but notCGM2. These CEA subfamily members share the B9 epitope in the N domain.These findings suggest that both mAbs are not CEA specific and mayindicate distinct functional properties of these different subfamilymembers.

A novel role for intestinal epithelial cells, that of antigen presentingcells, has been previously established: IECs can take up solubleproteins and present them to T cells. In the normal state suchpresentation results in the selective activation of CD8⁺ suppressor Tcells. This interaction has been further characterized by identifyingtwo molecules expressed by normal IECs which bind to the TcR and CD8molecules on these regulatory T cells, CD1d, and a molecule recognizedby two anti-epithelial mAbs previously generated by the inventors (B9and L12), which was referred to as gp180. Both mAbs recognizing gp180were initially identified in a functional screen. Purified gp180 wasshown to be capable of binding to CD8, activating p56 Ick and of bindingto CD1d.

In the foregoing examples, gp180 has been identified as CEA. This wasconfirmed by sequence analysis, the ability of B9 and L12 to stain CEAtransfectants and the ability of CEA to bind to CD8. It is intriguing tonote that both mAbs B9 and L12 recognize epitopes that are within 1AA ofeach other and are associated with a critical functional domain in theCEA molecule. Anti-CEA mAb B18 which recognizes an epitope furtherC-terminal fails to block p56 Ick activation in IEC: T cell co-cultures.These studies did not define the domain involved in CD1d complexformation. Since many CEA subfamily members can express the B9 epitope(the CD8 binding domain), their ability to activate distinct suppressorT cell subpopulations may depend upon their ability to bind to differentclass Ib molecules.

Despite the fact that CEA was identified in 1965 by Gold and Freeman,the functional relevance of this molecule and its associated subfamilymembers has been poorly understood. The most consistently describedfunction has been one of adhesion and this property has been suggestedto help explain its role in tumor metastasis. The inventors here havedescribed an important and heretofore unrecognized immunoregulatory rolefor CEA in a very specific fashion. It is intriguing to speculate thatas CEA expressing adenocarcinomas grow and enhance their expression ofCEA, the ability of this molecule to participate in the activation ofCD8⁺ suppressor T cells may allow for the tumor to escapeimmunologically mediated destruction. Another CEA subfamily memberCEACAM1 (BGP) is expressed on a number of other cell types including Tcells. CEACAM1 (BGP) has a transmembrane anchor and it contains an ITIMmotif in its intra-cytoplasmic trail. As such, binding of CEACAM1 maytransmit negative signals to cytotoxic T cells blocking their ability tokill targets. Since CEA subfamily members bind together by bothhomotypic and heterotypic interactions it is also conceivable that CEAexpressed by adenocarcinomas bind to CEACAM1 and CD8 delivering twonegative signals to the T cell; blocking cell mediated cytolysis andtumor killing.

EXAMPLE 5

gp180 Inhibits the Generation of CTLs but Fails to Inhibit TheirEffector Function.

The engagement of CD8 by gp180/CEA has been shown to utilizenonoverlapping sites to those used by MHC class I molecules. To addressthis more directly, increasing amounts of purified gp180 were added to aCTL assay testing alloreactive CTL (generated in mixed lymphocytereactions) activity against their target. As seen in FIG. 6 a, unlikemAbs to MHC class I or CD8α which both inhibited CTL activity,monoclonal antibody B9 affinity purified gp180 induced only a modestdecrease (<15% in 3 experiments) in killing, comparable to a CEA(non-monoclonal-antibody-B9-binding) control glyco-protein. In contrast(FIG. 6 b), when varying concentrations of purified gp180 were added tocultures where CTLs were induced, there was complete inhibition of thegeneration of CTLs. The inhibition was only seen when gp180/CEA wasadded to these cultures within the first 24 hours of co-culture.Addition of gp180 did not affect cell number or viability of the cellsin culture. These data indicate that gp180/CEA is altering early eventsinvolved in the generation of CTLs.

It was determined next whether similar effects could be seen inanti-viral CTL generation. PBMNC were infected with influenza strainBeijing and co-cultured with freshly isolated autologous peripheralblood T cells (PBT) in the presence or absence of varying concentrationsof gp180/CEA. These cultures were maintained for 5 days, at which timevirus activated T cells were incubated with influenza infected oruninfected autologous target cells (PHA stimulated T cell blasts).Consistent with the findings seen in FIG. 6, complete inhibition of CTLgeneration was seen when the priming cultures included gp180/CEA.

HT29 cells, a human colon cancer cell line, have been reported to be atarget for LAK cells. Thus this line can be killed by cytolytic T cells.HT29 constitutively expresses gp180/CEA. Therefore, it was determinedwhether HT29 cells could prime an anti-influenza CTL response. HT29cells were transfected with HLA-A2 using a retroviral construct. PB Tcells from an HLA-A2+ donor were co-cultured with either an influenzainfected or uninfected HLA-A2 expressing B cell line, autologous PBMC orthe A2 transfected HT-29 cells. Infection was confirmed in all cells byflow cytometry using an anti-HA specific mAb. After 5 days ofco-culture, T cells were incubated with autologous flu infected targetcells and killing was assessed. In the absence of influenza infection,no spontaneous killing was seen. Both the A2 expressing B cell line andautologous MNC primed for a CTL response whereas the A2 expressing HT29cells did not (FIG. 7 a). The absence of priming by the HT29 cells didnot necessarily reflect the expression of gp180/CEA alone. Intestinalepithelial cells fail to express a number of co-stimulatory moleculeswhich might be important in the generation of CTL. B7-1 (CD80) cDNA wastherefore transfected into HT29 HLA-A2 transfectants and their abilityto prime a flu specific CTL response was determined. As seen in FIG. 7 bwhile there was restoration of CTL activity there was no evidence of Agspecificity. HLA-A2+ T cells killed both the infected and noninfectedcells with similar efficiency. These findings were consistent with theactivation of LAK cells rather than class I restricted CTLs.

EXAMPLE 7

gp180/CEA Alters the Signaling Pathway within CTLs.

Since gp180/CEA does not appear to inhibit the ability of CD8 to engageclass I molecules, the mechanism underlying the inhibition seen wasinvestigated. Previous studies by the present inventors had shown thatengagement of CD8 by gp180/CEA results in the rapid autophosphorylationof the CD8 associated src-like kinase p561ck but not the TcR associatedkinase p59fyn. In contrast, intact epithelial cells co-cultured with PBT cells induce phosphorylation of both Ick and fyn, reflecting theengagement of the TcR by CD1d and CD8 by gp180/CEA. Downstreamsubstrates and kinases are phosphorylated as well including theguanosine exchange factor Vav which is phosphorylated rapidly aftergp180/CEA: T cell co-culture. Previous studies have documented asignificant role for PI3K and PLCγ1 as well as Vav in the activation ofclass I restricted CTLs. Given the rapid kinetics of Vav phosphorylationin either IEC: T cell co-cultures or in T cell: gp180/CEA co-cultures,it was determined whether there was an alteration in signaling thatwould account for gp180/CEA's ability to inhibit CTL generation. As seenin FIG. 8 a co-culture of PB T cells with allogeneic adherent cells(DCs, monocytes) resulted in both phosphorylation of PI3K and Vav (andPLCγ1-not shown). In contrast, co-culture of freshly isolated IECs withthese same T cells failed to induce PI3K phosphorylation but did inducethe phosphorylation of Vav. Confirmation of the role of PI3K in CTLactivation came in studies using wortmanin in these same co-cultures.This PI3K inhibitor blocked the generation of alloreactive CTLs butfailed to block proliferation of T cells activated by allogeneicepithelial cells. Given these findings, it was necessary to determinewhether gp180/CEA had the capacity to alter P13K phosphorylation. Asseen in FIG. 8 b, addition of gp180/CEA to T cell: allogeneic adherentcell co-cultures resulted in the loss of PI3K phosphorylation and abrisk upregulation of phospho-Vav. These data indicate that engagementof CD8 by gp180 (CEA) has the capacity to redirect intracellularsignaling pathways which may be important in CTL generation.

By the very nature of its co-existence with a sea of antigens one celllayer away from the largest lymphoid organ in the body, the mucosal(intestinal) immune system requires distinct mechanisms to induce andcontrol immune responses. In general, the immunologic tone of the GItract is one of tight regulation and suppression of immune responses. Avariety of factors are likely to be involved in this process. Thelymphoid populations present in the GI tract are unique, being resistantto signals mediated through the antigen receptor and exquisitelyresponsive to co-stimulatory pathways. Professional APCs in the mucosalack conventional receptors (e.g. CD14) and their ability to stimulateimmune responses is modest. Given the location of the intestinalepithelial cells between the antigen-laden intestinal lumen and the cellrich lamina propria, several groups have proposed a novel function forthese cells, that of nonprofessional APC. Indeed various investigatorshave documented the ability of IECs to either activate class IIrestricted memory cells or class lb restricted suppressor/regulatory Tcells. These latter cells are CD8+ CD28^(+ or −) IL-2R⁺ and for the mostpart appear to recognize CD1d as their restriction element. The natureof the antigen presented by this class lb molecule has not as yet beendefined. The activation of these class lb restricted suppressor cellshas been shown to be dependent upon the activation of protein tyrosinekinases including p561ck and p59fyn. However, the expression of CD1dalone does not totally account for activation of these cells. Amolecular chaperone, a membrane glycoprotein called gp180, is a keycomponent of these interactions. gp180/CEA binds to CD8 and activatesCD8 associated p561ck. mAbs to gp180/CEA (mAb B9 and L12) block theproliferation and 1ck activation in T cells induced by IECs.

In the foregoing examples, the role of gp180/CEA was assessed in themodulation of responses which might promote inflammation. In thiscontext it is interesting to note that, while enteric viral infectionsoccur in class I-expressing IECs, generalized cytolysis of theepithelium or even selected areas of ulceration do not occur in thecourse of these infections. This finding documents that gp180/CEA iscapable of inhibiting CTL generation without affecting cell viability ornumbers. However, the expression of gp180/CEA by itself does not rendertargets resistant to lysis. Therefore, were anti-viral CTLs to begenerated elsewhere, there is potential for those cells to lyseepithelial targets.

The effect of gp180/CEA on CTLs may be only one of several mechanismswhereby this molecule participates in maintaining controlledinflammation or immunological unresponsiveness in the gut. As alluded toearlier, gp180/CEA is a key component in the generation of class lbrestricted regulatory T cells. Activation of these cells would addfurther to controlled inflammation.

The mechanism whereby gp180/CEA blocks CTL generation is also ofinterest. Previous studies have defined a post receptor pathway for allcytolytic cells which includes PI3K and Vav. By mechanisms which remainto be defined, co-incubation of T cells with gp180/CEA results in theabsence of PI3K phosphorylation and the rapid and robust phosphorylationof Vav. This appears to correlate with the alteration in T cellfunction. Such a phenomenon is not without precedent as the presence orabsence of other co-stimulatory molecules (e.g. B7-1, B7-2) haveprofound effects on the nature of the T cell response. Interestinglydespite the fact that both class I MHC and gp180/CEA bind to CD8 (albeitat different sites), signals transduced by these two molecules are quitedifferent.

In summary, the foregoing studies have demonstrated that the epithelialglycoprotein gp180, also known as CEA, has the capacity to regulatepotentially harmful immune responses. Such a finding underscores thepotential role for this molecule in regulating mucosal immune responses.Taken together with clinical observations of the failure of anendogenous cellular immune response to be mountable against antigens orepitopes expressed or over-expressed in certain tumors, the poor immuneresponse elicited when full-length CEA is utilized as an immunogen, andthe phenomenon of oral tolerance, the central immunomodulatory role ofCEA in these and other instances is clearly a target for modulation inorder to overcome the cellular immunosuppression often seen andresponsible for failures in an otherwise promising immunotherapeuticapproach to the treatment of cancer, as well and in prevention ofcertain infectious diseases.

The present invention is not to be limited in scope by the specificembodiments describe herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Various publications are cited herein, the disclosures of which areincorporated herein by reference in their entireties.

1. A method for effectively immunizing an animal against a tumorexpressing a carcinoembryonic antigen comprising immunizing said animalusing an immunogen consisting of said carcinoembryonic antigen modifiedto lack cellular immunosuppressive activity or CD1d binding.
 2. Themethod of claim 1 wherein said carcinoembryonic antigen modified to lackcellular immunosuppressive activity comprises said carcinoembryonicantigen having a region recognized by monoclonal antibody B9 or L12modified to inactivate said cellular immunosuppressive activity.
 3. Themethod of claim 1 wherein said region is SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3.
 4. The method of claim 1 wherein said immunogen comprises adeletion, alteration, mutation, or truncation of said cellularimmunosuppressive region.
 5. The method of claim 1 wherein saidimmunogen is an N domain deletion, a K35A substitution in the N domain,or a deletion of GYSWYK or NRQII.
 6. A method for effectively immunizingan animal against a carcinoembryonic-antigen-expressing tumor comprisingimmunizing said animal using an immunogen consisting of saidcarcinoembryonic antigen modified to lack an active cellularimmunosuppressive region.
 7. The method of claim 6 wherein said cellularimmunosuppressive region comprises a monoclonal antibody B9- orL12-recognizing region.
 8. The method of claim 6 wherein said activecellular immunosuppressive region is SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3.
 9. The method of claim 6 wherein said immunogen comprises adeletion, alteration, mutation, or truncation of said active cellularimmunosuppressive region.
 10. The method of claim 6 wherein saidimmunogen is a N domain deletion, or a K35A substitution in the Ndomain, or a deletion of GYSWYK or NRQII.
 11. A method for effectivelyimmunizing an animal against a carcinoembryonic-antigen-expressing tumorcomprising immunizing said animal using an immunogen consisting of saidcarcinoembryonic antigen having a K35A substitution in the N domain, adeletion of GYSWYK or a deletion of NRQII.
 12. A method for enhancingcellular immunogenicity of an orally-delivered immunogen in an animalcomprising co-administering with said orally-delivered immunogen anagent capable of inhibiting the cellular immunosuppressive activity ofcarcinoembryonic antigen.
 13. The method of claim 12 wherein said agentis a ligand of said carcinoembryonic antigen capable of disrupting theengagement of said carcinoembryonic antigen family member with CD8,disrupting the association of said carcinoembryonic antigen familymember with CD1d, or the combination of both.
 14. The method of claim 13wherein said ligand is a monoclonal antibody.
 15. The method of claim 14wherein said monoclonal antibody is B9 or L12.
 16. The method of claim12 wherein said agent is an inhibitor of the engagement of CD8 with acellular immunosuppressive region of said carcinoembryonic antigenfamily member, or an inhibitor of the engagement of CD1d with a cellularimmunosuppressive region of said carcinoembryonic antigen family member.17. The method of claim 16 wherein said agent is a CD8-Fc fusion peptideor a CD8 peptide comprising an epitope recognized by any one ofantibodies OKT8B, OKT8E, OKT8F or OKT8I1.
 18. A method for treating apatient having a carcinoembryonic-antigen-family-member-expressing tumorcomprising immunizing said patient with an immunogen consisting of saidcarcinoembryonic antigen family member modified to lack a cellularimmunosuppressive region.
 19. The method of claim 19 wherein saidcarcinoembryonic antigen family member is lacking a B9/L12 epitope. 20.A method for suppressing a humoral or cellular immune response in amammal to an antigen comprising administering to a site of said humoralor cellular response in said mammal an agent comprising a cellularimmunosuppressive region of a carcinoembryonic antigen family member.21. The method of claim 20 wherein said agent comprising a cellularimmunosuppressive region of a carcinoembryonic antigen family member iscarcinoembryonic antigen, a carcinoembryonic antigen family member, or afragment or fusion polypeptide of either of the foregoing comprising acellular immunosuppressive region.
 22. The method of claim 20 whereinsaid administering is introducing a vector capable of inducingexpression of said agent in cells of said mammal.
 23. The method ofclaim 20 wherein said mammal is a human.
 24. The method of claim 20wherein said carcinoembryonic antigen family member is carcinoembryonicantigen.
 25. A pharmaceutical composition for eliciting an effectiveimmune response to a carcinoembryonic antigen family member comprisingan immunogen consisting of said carcinoembryonic antigen family membermodified to lack an active cellular immunosuppressive region.
 26. Thepharmaceutical composition of claim 25 wherein said cellularimmunosuppressive region comprises a monoclonal antibody B9- orL12-recognizing region.
 27. The pharmaceutical composition of claim 25wherein said active cellular immunosuppressive region is SEQ ID NO:1,SEQ ID NO:2, or SEQ ID NO:3.
 28. The pharmaceutical composition of claim25 wherein said immunogen comprises a deletion, alteration, mutation, ortruncation of said active cellular immunosuppressive region.
 29. Thepharmaceutical composition of claim 28 wherein said immunogen is a Ndomain deletion, or a K35A substitution in the N domain, or a deletionof GYSWYK or NRQII.
 30. The pharmaceutical composition of claim 25wherein said carcinoembryonic antigen family member is carcinoembryonicantigen.